The invention relates to a process for preparing thrombin-like proteolytic enzymes from venoms of snakes, particularly snakes of the family Crotalidae, especially of the genera Agkistrodon, Bothrops, Crotalus and Trimeresursu.
Thrombin-like proteolytic enzymes are proteases which are obtained for instance from certain snake venoms and which, like thrombin, split fibrinopeptide A and/or fibrinopeptide B from fibrinogen, but which, unlike thrombin, have a high substrate specificity [cf. e.g. D. L. Aronson, Thrombos. Haemostas. (Stuttg.), 1976, 36, pages 9 to 13, and 1976, 35, page 477].
Thrombin-like snake venom enzymes are used as reagents for investigating blood coagulation processes, as antihaemorrhagic drugs and as agents for the experimental and therapeutic defibrinogenation and, hence, anticoagulation.
The following Table comprises indications relating to the use of thrombin-like enzymes from the venoms of some snake species and the respective literature references.
TABLE 1 ______________________________________ Venom from the species: Use for: Literature: ______________________________________ Agkistrodon blood tests R.M. Herzig, O.D. Ratnoff +-contortrix Y.R. Shainoff: Studies on a procoagulant fraction of southern copperhead snake venom: The preferential re- lease of fibrinoipeptide B. J. Lab. and Clin. Med. 76, 451-465 (1970) Agkistrodon defibrino- A.A. Sharp, B.A. Warren, rhodostoma genation A.M. Paxton + M.J. Allington: Anticoagulant therapy with a purified fraction from Malayan pit viper venom. Lancet I, 493-499 (1968) Bothrops atrox defibrino- N. Egberg: Experimental and genation clinical studies on the thrombin-like enzyme from the venom of Bothrops atrox. On the primary structure of fragment E. Acta phys. scand. Suppl. 400 (1973) haemostasis E. Berger, A.J. Laurent + K.F. Stocker: The prophyl- actic and therapeutic use of Reptilase. Praxis 57, No. 17, 611-616 (1968) Blood tests C. Funk, J. Gmur, R. Herold + P.W. Straub: Reptilase-R. A new reagent in blood co- agulation. Brit. J. Haematol. 21, 43- 52 (1971) Bothrops haemostasis C. Mauro: Sulla azione emo- jararaca coagulante del veleno di Bothrops jararaca. Giorn.Ital. di Chirurgia 8, 448 (1949) Crotalus defibrino- F.S. Markland + P.S. Damus: adamanteus genation Purification and properties from the venom of Crotalus adamanteus. J. Biol. Chem. 246, 6460- 6473 (1971) Trimeresurus defibrino- C. Ouyang + F.Y. Yang: gramineus genation Purification and properties of the thrombin-like enzyme from Trimeresurus grami- neus venom. Biochim. et Biophys. Acta 351, 345-363 (1974). ______________________________________
The said proteolytic enzymes are glycopeptides having molecular weights comprised between 18,000 and 55,000. Since they are inhibited by di-isopropyl fluorophosphate, they have to be classified in the serine protease group. Like thrombin, the thrombin-like snake venom enzymes cause the conversion of fibrinogen into fibrin by the release of fibrinopeptides; however, they differ from thrombin in their behaviour towards other blood coagulation factors and thrombocytes, and, more particularly, by the fact that their coagulating activity on plasma is not significantly inhibited by thrombin inhibitors such as heparin, hirudin, antithrombin III and heparinoids [cf. D. L. Aronson, Thrombos. Haemostas. (Stuttg.), 1976, 36, pages 9 to 13].
In order to concentrate the thrombin-like enzymes from snake venoms which generally consist of mixtures of more than 20 different pharmacologically active polypeptides, DEUTSCH (cf. E. Deutsch, "Blutgerinnungsfaktoren," Verlag Deuticke, Vienna 1955) precipitated impurities from dissolved crude venom by the action of acids and heat and recovered the thrombin-like enzymes as an enriched fraction from the remaining liquid phase by precipitation with solvents or salts. BANERJEE et al. have described the thirty-fold concentration of the thrombin-like enzyme from venom of Bothrops jararaca by ammonium sulfate precipitation from a 0.5% solution of crude venom, heat treatment of the dissolved precipitate at 65.degree. C., separation of the precipitated impurities by centrifugation, absorption of the thrombin-like enzyme on calcium phosphate gel, elution with sodium phosphate buffer at pH 7.2, repeated fractional ammonium sulfate precipitation and finally dialysis against veronal buffer [cf. E. Banerjee, A. Devi + N. Sarkar: Isolation and purification of a coagulant from snake venom of the species Bothrops jararaca and the study of its properties, Thromb. Diath. Haem. 5, 296-303 (1960)]. British patent specifications 1,094,301 and 1,177,506 disclose the preparation of the thrombin-like enzyme from the venom of Agkistrodon rhodostoma by chromatography on triethylaminoethylcellulose and subsequent purification by gel chromatography. Thus, 2 g of crude Agkistrodon rhodostoma venom are subjected to chromatography first on a TEAE-cellulose column of 35 .times. 3.9 cm (= 417 ml) and then on a "Sephadex G-100" column of 97 .times.6 cm (= 2826 ml). BONILLA [Thromb. Res. 6, 151-169 (1975)] describes the isolation of the thrombin -like enzyme from the venom of Crotalus horridus by chromatography of 20 g of crude venom on a column of 1766 ml of "Sephadex G-100," subsequent chromatography of the active fraction on a column of 883 ml of diaminoethylcellulose, a further chromatography on 883 ml of carboxymethylcellulose and a last purification by chromatography on a DEAE-cellulose column of 2.5 .times. 36 cm (176 ml) using a linearly increasing buffer concentration gradient and subsequent chromatography on a "Sephadex G-200" column of 2.5 .times. 33 cm. While the methods based on ion exchange and gel chromatography allow a much higher purity to be obtained than the first described adsorption method, they are time-consuming and uneconomic since relatively large columns have to be used for the chromatography of only small quantities of crude venom. Furthermore, the active eluate contains a relatively small quantity of enzyme in a large volume of liquid and, therefore, has to be concentrated either by ultrafiltration or vacuum distillation. According to U.S. Pat. No. 3,849,252 the thrombin-like enzyme from Bothrops atrox venom is obtained by precipitating impurities from 20 g of crude venom by acidification, then precipitating the enzyme by formation of a sparingly soluble complex with phenol or a phenol derivative, decomposing the said complex, subjecting the enzyme thus concentrated to a chromatography on a column of 200 ml of DEAE-Sephadex and thereafter purifying the product on a "Sephadex G-100" column of 1.8 .times. 92 cm (233 ml). Due to the precipitation of the thrombin-like enzyme as a complex with phenol or a phenol derivative the output of the chromatographic purification steps of this method is substantially higher than in the direct chromatography of the crude venom; however, the numerous steps of this method require a considerable expenditure of work and yield a product which has an insufficient purity and which forms with prothrombin-free plasma in the presence of calcium ions a clot insoluble in monochloroacetic acid and thus activates factor XIII. HOLLEMANN and WEISS [J. Biol. Chem. 251, 1663-1669 (1976)]achieved the isolation and purification of thrombin-like enzyme from Bothrops atrox venom by affinity chromatography of 2 g of crude venom on a 2.5 .times. 56 cm column (274 ml) of p-aminobenzamidinsuccinyl-diaminodipropylaminoagarose at 4.degree. C. using 0.15 mole of benzamidine in a sodium citrate/NaCl buffer having a pH of 9.0 and subsequent removal of the benzamidine from the eluate by dialysis. In this manner they obtained a product which did not activate factor VIII. This method, although yielding a product of sufficient purity in a few steps only, requires a relatively large column of an adsorbent which is difficult to prepare and, moreover, necessitates the use, as an elution agent, of benzamidine which, owing to its property to absorb ultraviolet light, prevents the UV-photometric control of the course of the chromatography and which, since it has a considerable toxicity, must be completely removed from the eluates, thus requiring a time-consuming dialysis.
It was now found that thrombin-like enzymes from snake venoms, although their clotting activity is not inhibited by heparin under the usual conditions of pH, ionic strength and temperature normally used in blood clotting tests, quite unexpectedly have a pronounced affinity for heparin which has been insolubilized by fixation on an insoluble carrier and can, therefore, be isolated from crude snake venoms or fractions thereof and purified by affinity chromatography on insolubilized heparin.